T cells expressing multiple co-inhibitory molecules in acute malaria are not exhausted but exert a suppressive function in mice.

Overwhelming activation of T cells in acute malaria is associated with severe outcomes. Thus, counter-regulation by anti-inflammatory mechanisms is indispensable for an optimal resolution of disease. Using Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice, we performed a comprehensive analysis of co-inhibitory molecules expressed on CD4+ and CD8+ T cells using an unbiased cluster analysis approach. We identified similar T cell clusters co-expressing several co-inhibitory molecules like programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) in the CD4+ and the CD8+ T cell compartment. Interestingly, despite expressing co-inhibitory molecules, which are associated with T cell exhaustion in chronic settings, these T cells were more functional compared to activated T cells that were negative for co-inhibitory molecules. However, T cells expressing high levels of PD-1 and LAG-3 also conferred suppressive capacity and thus resembled type I regulatory T cells. To our knowledge, this is the first description of malaria-induced CD8+ T cells with suppressive capacity. Importantly, we found an induction of T cells with a similar co-inhibitory rich phenotype in Plasmodium falciparum-infected patients. In conclusion, we demonstrate that malaria-induced T cells expressing co-inhibitory molecules are not exhausted, but acquire additional suppressive capacity, which might represent an immune regulatory pathway to prevent further activation of T cells during acute malaria.

OMIP-93: A 41-color high parameter panel to characterize various co-inhibitory molecules and their ligands in the lymphoid and myeloid compartment in mice.

This 41-color panel has been designed to characterize both the lymphoid and the myeloid compartments in mice. The number of immune cells isolated from organs is often low, whilst an increasing number of factors need to be analyzed to gain a deeper understanding of the complexity of an immune response. With a focus on T cells, their activation and differentiation status, as well as their expression of several co-inhibitory and effector molecules, this panel also allows the analysis of ligands to these co-inhibitory molecules on antigen-presenting cells. This panel enables deep phenotypic characterization of CD4+ and CD8+ T cells, regulatory T cells, γδ T cells, NK T cells, B cells, NK cells, monocytes, macrophages, dendritic cells, and neutrophils. Whilst previous panels have focused on these topics individually, this is the first panel to enable simultaneous analysis of these compartments, thus enabling a comprehensive analysis with a limited number of immune cells/sample size. This panel is designed to analyze and compare the immune response in different mouse models of infectious diseases, but can also be extended to other disease models, for example tumors or autoimmune diseases. Here, we apply this panel to C57BL/6 mice infected with Plasmodium berghei ANKA, a mouse model of cerebral malaria.

Pathogenetic mechanisms and treatment targets in cerebral malaria.

Malaria, the most prevalent mosquito-borne infectious disease worldwide, has accompanied humanity for millennia and remains an important public health issue despite advances in its prevention and treatment. Most infections are asymptomatic, but a small percentage of individuals with a heavy parasite burden develop severe malaria, a group of clinical syndromes attributable to organ dysfunction. Cerebral malaria is an infrequent but life-threatening complication of severe malaria that presents as an acute cerebrovascular encephalopathy characterized by unarousable coma. Despite effective antiparasite drug treatment, 20% of patients with cerebral malaria die from this disease, and many survivors of cerebral malaria have neurocognitive impairment. Thus, an important unmet clinical need is to rapidly identify people with malaria who are at risk of developing cerebral malaria and to develop preventive, adjunctive and neuroprotective treatments for cerebral malaria. This Review describes important advances in the understanding of cerebral malaria over the past two decades and discusses how these mechanistic insights could be translated into new therapies.

Increased Expression of Multiple Co-Inhibitory Molecules on Malaria-Induced CD8+ T Cells Are Associated With Increased Function Instead of Exhaustion.

Activated cytotoxic CD8+ T cells can selectively kill target cells in an antigen-specific manner. However, their prolonged activation often has detrimental effects on tissue homeostasis and function. Indeed, overwhelming cytotoxic activity of CD8+ T cells can drive immunopathology, and therefore, the extent and duration of CD8+ T cell effector function needs to be tightly regulated. One way to regulate CD8+ T cell function is their suppression through engagement of co-inhibitory molecules to their cognate ligands (e.g., LAG-3, PD-1, TIM-3, TIGIT and CTLA-4). During chronic antigen exposure, the expression of co-inhibitory molecules is associated with a loss of T cell function, termed T cell exhaustion and blockade of co-inhibitory pathways often restores T cell function. We addressed the effect of co-inhibitory molecule expression on CD8+ T cell function during acute antigen exposure using experimental malaria. To this end, we infected OT-I mice with a transgenic P. berghei ANKA strain that expresses ovalbumin (PbTG), which enables the characterization of antigen-specific CD8+ T cell responses. We then compared antigen-specific CD8+ T cell populations expressing different levels of the co-inhibitory molecules. High expression of LAG-3 correlated with high expression of PD-1, TIGIT, TIM-3 and CTLA-4. Contrary to what has been described during chronic antigen exposure, antigen-specific CD8+ T cells with the highest expression of LAG-3 appeared to be fully functional during acute malaria. We evaluated this by measuring IFN-γ, Granzyme B and Perforin production and confirmed the results by employing a newly developed T cell cytotoxicity assay. We found that LAG-3high CD8+ T cells are more cytotoxic than LAG-3low or activated but LAG-3neg CD8+ T cells. In conclusion, our data imply that expression of co-inhibitory molecules in acute malaria is not necessarily associated with functional exhaustion but may be associated with an overwhelming T cell activation. Taken together, our findings shed new light on the induction of co-inhibitory molecules during acute T cell activation with ramifications for immunomodulatory therapies targeting these molecules in acute infectious diseases.